Abstract

To gain insights into microgravity-induced ophthalmic changes (microgravity ocular syndrome), and as part of a project investigating effects of future planetary habitats, we investigated the effect of acute hypercapnia following 10-day bed rest and hypoxia on posterior eye structures. Female subjects (N = 7) completed three 10-day experimental interventions: 1) normoxic bed rest [NBR; partial pressure of inspired O2 (PiO2) = 132.9 ± 0.3 Torr]; 2) hypoxic ambulatory confinement (HAMB; PiO2 = 90.4 ± 0.3 Torr); and 3) hypoxic bed rest (HBR; n = 12; PiO2 = 90.4 ± 0.3 Torr). Before and on the last day of each intervention, optical coherence tomography (OCT) of the optic disk was performed, and the thicknesses of the retinal nerve fiber layer (RNFL), retina, and choroid were measured. OCT examinations were conducted with the subjects breathing the prevailing normocapnic breathing mixture (either normoxic or hypoxic) and then following a 10-min period of breathing the same gas mixture, but with the addition of 1% CO2. Choroidal thickness was greater during both bed-rest conditions (NBR and HBR) compared with the ambulatory (HAMB) condition (ANOVA, P < 0.001). Increases in RNFL thickness compared with baseline were observed in the hypoxic trials (HBR, P < 0.001; and HAMB, P = 0.021), but not the normoxic trial (NBR). A further increase in RNFL thickness (P = 0.019) was observed after the 10-min hypercapnic trial in the NBR condition only. The fact that choroidal thickness was not affected by Po2 or Pco2, but increased by bed rest, suggests a hydrostatic rather than a vasoactive effect. The increments in RNFL thickness were most likely associated with local hypoxia and hypercapnia-induced dilatation of the retinal blood vessels.

space life sciences

planetary habitats

ophthalmology

hypercapnia

hypoxia

NEW & NOTEWORTHY

Horizontal 10-day bed rest, followed by acute hypercapnia and hypoxia, resulted in morphological changes of posterior eye structures measured by optical coherence tomography. Bed rest caused an increase in the thickness of the choroidal layer. Hypoxia and hypercapnia caused an increase in the thickness of the retinal nerve fiber layer (RNFL). It appears that the increase in the choroidal layer is affected predominantly by a hydrostatic, and the RNFL by a vasoactive, effect.

the term microgravity ocular syndrome (MOS) has been used to describe the structural and functional changes documented initially in seven astronauts after long-duration spaceflight (19). The structural changes associated with this syndrome include various degrees of optic disk edema, choroidal folds, flattening of the globe, and retinal nerve fiber ischemia (cotton wool spots). These changes usually remain below the functional threshold, with the exception of hyperopic (far-sighted) shift. Before the discovery of MOS, anecdotal reports of astronauts commented on vision changes following short- and long-duration spaceflight. These included primarily hyperopic shifts, resulting in reduction of near visual acuity, which was attributed to the age of the astronauts. The etiology of MOS remains unresolved (19, 20, 32).

Bed rest results in inactivity and unloading of weight-bearing muscles and bones, and the bed rest experimental analog has been successfully used to investigate the effects of microgravity on several physiological systems (25). However, the effect of bed rest on the structure and function of the eye has been largely ignored. Most previous studies have focused on the intraocular pressure (IOP) (2, 5, 15), but have yielded equivocal results. Ocular changes, if any, were subclinical, and did not significantly alter visual function (15, 33). A more recent study utilizing optical coherence tomography (OCT), an imaging modality used to acquire in vivo cross-sectional images of the retina, reported an intriguing finding: there was a subtle increase in retinal thickness in the area around the optic disk (i.e., peripapillary region) after 30 days of 6° head-down-tilt bed rest compared with matched baseline measurements conducted 6 mo after the bed rest (32). Subsequently, Taibbi et al. (33) reported an increase in retinal nerve fiber layer (RNFL) thickness, but not in total peripapillary retinal thickness, after 10 days of head-down-tilt bed rest. To date, the effect of prolonged bed rest on the choroid has not been studied. The choroid is the middle, vascular coat of the eye, between the sclera and the retina, and contains an abundant supply of blood vessels. Seventy percent of the blood flow to the eye is distributed to the choroid (23). One main function is to supply oxygen and metabolites to the outer retina, retinal pigment epithelium (RPE), and possibly the prelaminar portion of the optic nerve (14), while the inner retina is perfused through the retinal vessels whose larger portion lay in RNFL. Notably, Shinojima et al. (30) observed increased thickness of the subfoveal choroid after acute (15 min) head-down tilt, which was likely caused by the increased hydrostatic pressure, as a consequence of the altered gravity vector.

Future long-term missions to the moon and Mars will require astronauts to live in habitats, in which the environmental conditions will be hypobaric and hypoxic, to minimize the risk of decompression sickness during preparation for extra-vehicular activities. Such environments are known to be conducive to the development of altitude retinopathy, as experienced by alpinists during high-altitude expeditions. Despite the fact that the microgravity-induced changes in retinal structure and concomitant deterioration in visual function remain unresolved, we hypothesized that the hypoxia anticipated in future lunar and planetary habitats may exacerbate these changes. An additional factor, which could be implicated in the observed changes, is hypercapnia. Although future habitats will incorporate systems for the removal of carbon dioxide (CO2 scrubbers) from the environment, current experience on the International Space Station (ISS) is that these systems are not capable of maintaining CO2 concentration at levels present on Earth. Indeed, Taylor et al. (34) reported that the partial pressure of CO2 within the ISS attained 3.5 Torr, which is more than 10-fold normal air levels on Earth. Currently, the space maximum allowable concentrations of CO2 on the ISS range from 0.5% (3.8 mmHg) for durations of 1,000 days, 0.7% for up to 180 days, 1.3% for 24 h, and 2% for 1 h (18). It is, therefore, imperative to consider the effect of such hypercapnia alone (as on the ISS) and in combination with hypoxia (as in future lunar and planetary habitats) on visual function.

The European Space Agency (ESA) and the Swedish National Space Board initiated a program of research to assess the potential effects of future hypobaric hypoxic environments on astronauts (6). This study is part of one such project investigating the effect of a 10-day hypoxic bed rest (HBR) on female subjects. The present report focuses on the effect of hypoxia in combination with hypercapnia on the structures of the posterior part of the eye, in particular the retina and the choroid.

METHODS

The study was part of a larger research program funded by ESA and the Swedish National Space Board to investigate the physiological effects of simulated planetary habitation on healthy female subjects. The study was conducted in the hypoxic facility at the Olympic Sport Centre Planica (Rateče, Slovenia), situated at an altitude of 940 m. The study protocol was approved by the National Committee for Medical Ethics at the Ministry of Health of the Republic of Slovenia. All experimental procedures were conducted in accordance with the ESA recommendations for bed-rest studies and conformed to the principles of the Helsinki Declaration.

Participants.

Seven healthy, nonsmoking eumenorrheic women participated in the present study. They passed the inclusion/exclusion criteria, which were based on the ESA recommendations for bed-rest protocols (standardization of bed rest study conditions, August 2009). They were all low-landers and had not been exposed to altitudes of 2,000 m or above within 2 mo before the start. Exclusion criteria also included chronic diseases and history of eye problems. All subjects had unremarkable ophthalmological examination at baseline. Two of the subjects were myopic (short-sighted) with refractive error of −1.50 and −3.25 diopters, respectively. Participation in the study was subject to physicians' approval, and before entering the study the participants provided informed consent and were given detailed information regarding the study protocol and all experimental procedures. The short-listed participants were also invited to the Olympic Sport Centre for a familiarization day, several weeks before the start of the study. Subjects' average ± SD baseline physical characteristics (age, body mass, stature, body fat, maximal oxygen uptake) are presented in Table 1.

The three experimental interventions were conducted concurrently within three experimental campaigns. During each experimental campaign, one part of the facility was rendered hypoxic, and the other part normoxic. Since the rooms in the facility were arranged to accommodate two subjects, subjects were allowed to choose their roommates during a familiarization weekend conducted at the facility several weeks before the start of the first experimental campaign. Before the first experimental campaign, subject pairs were randomly assigned to one of the three interventions: NBR, HBR, and HAMB. For each of the subject pairs, the order of the other two interventions conducted in the remaining two experimental campaigns was assigned according to a Latin square design.

Campaigns lasted 19 days for each individual participant and had three phases. 1) Preintervention phase: This phase allowed the participants to acclimate to the facility, diet, and circadian cycles. All baseline (Pre) measures were obtained during this period. 2) Intervention phase: This was a 10-day (240 h) confinement phase during which the participants were exposed to the designated condition (NBR, HAMB and HBR). 3) Postintervention phase: Following the 10-day intervention, subjects remained at the facility for a 4-day recovery phase that enabled the researchers to obtain the postconfinement measurements (Post) and allowed for cautious reambulation of the participants. The campaigns were separated by a minimum period of 1 mo to enable sufficient physiological and psychological recovery of the participants.

Bed rest and hypoxic ambulation protocol.

A high-fidelity ground-based simulation of a lunar habitat would require a 9° head-up tilt during the day, to simulate the vascular pressure gradients due to the gravity on the moon, and a horizontal bed rest at night. However, since the priority of this study was a fundamental assessment of the separate and combined effects of inactivity/unloading and hypoxia on physiological systems, the participants were confined to strict horizontal bed rest during the NBR and HBR interventions. Horizontal bed rest is a regularly employed ground-based model to simulate microgravity-induced unloading (25). The participants were accommodated in rooms with two single beds (i.e., two participants per room). To standardize the circadian rhythm, the participants were awakened daily at 7:00 AM, and room lights were turned off at 11:00 PM. Sleeping was not allowed during the daytime hours. Morning resting heart rate (HR) and capillary oxyhemoglobin saturation (SpO2) were measured daily using short-range telemetry (iBody, Wahoo Fitness, Atlanta, GA) and finger oximetry (3100 WristOx, Nonin Medicals), respectively. To assess the potential presence and, where relevant, severity of altitude mountain sickness (AMS), the participants filled out the self-assessment part of the Lake Louise AMS questionnaire every evening at 8:00 PM throughout the confinement period (28). The Lake Louise Score (0-15) was subsequently calculated by summing the values of the individual questionnaire items. Both of the following criteria had to be fulfilled to diagnose AMS: 1) Lake Louise Score ≥ 3; and 2) presence of headache. All daily activities (i.e., reading, watching television, showering, lavatory) were carried out in the horizontal position. The participants were allowed to use one pillow for head support. No physical activity, apart from changing positions from supine, prone, and lateral, was permitted during the bed-rest confinement phase. Compliance to the bed-rest protocol was ensured using continuous closed-circuit television monitoring as well as permanent 24/7 medical staff supervision. To ease the neck- or backache that often occurs during the initial 2–3 days of the bed rest, the participants were provided, on request, with mild analgesic (acetaminophen) and/or passive stretching performed by a certified physiotherapist.

During the HAMB confinement, the participants were encouraged to engage in their habitual routines and allowed to move freely in the common hypoxic area (110-m2 surface area). Throughout the HAMB confinement, the participants also performed low-intensity exercise sessions to mimic their habitual physical activity. Two 30-min sessions were performed daily: one in the morning and one in the afternoon. The exercise mode (stepping, cycling, or dancing) was rotated to avoid monotony. During all exercise sessions, short-range telemetry and finger oximetry were employed to ensure that the individual's HR was within the targeted range, and to monitor the SpO2 levels, respectively. Assessment of the target exercise intensity, and thus energy expenditure levels, was based on the daily physical activity questionnaires filled out by the participants before the study. On average, the prescribed exercise induced a HR corresponding to that achieved at 50% of the peak power output (PPO), determined during a hypoxic graded exercise test. The graded test was performed before the HAMB confinement on a cycle ergometer under hypoxic conditions (FiO2 = 0.144) using 25 W/min workload increments until task failure (inability to maintain cycling cadence > 60 rpm). PPO was calculated according to the following formula: PPO (Watts) = Wcomp + [t × (60 × 30)−1], where Wcomp is last completed workload, and t is seconds during the final uncompleted workload.

In normoxic ground-based simulations using the bed rest experimental model, maximum aerobic capacity is assessed before and immediately after the cessation of the bed rest, to assess the degree of bed rest-induced cardiovascular deconditioning. This was also the principal reason that a normoxic maximal aerobic capacity test was conducted at these time points in the present study. To establish whether adaptive effects were condition specific, the subjects also conducted an aerobic capacity test under hypoxic conditions, Thus, due to an anticipated increase in erythropoiesis in the hypoxic confinement, and the concomitant increase in erythrocyte mass, it was considered possible that this might attenuate the bed-rest-induced decrease in aerobic capacity in the HBR confinement. This was one of the reasons for conducting a hypoxic aerobic capacity test before and after HBR. The second reason was to be able to assign an appropriate absolute workload for the daily exercises in the HAMB confinement.

Hypoxic facility.

The normobaric hypoxic environment was established and maintained using a vacuum pressure swing adsorption system (b-Cat, Tiel, The Netherlands) that delivered the O2-depleted gas to the designated rooms and common hypoxic area. The ambient air in each room was sampled and analyzed for O2 and CO2 content at 15-min intervals. Before the analysis of the air samples, the O2 and CO2 analyzers were automatically calibrated using precision calibration gases (Messer, Ljubljana, Slovenia). Immediate adjustments were made in the event of a >0.5% variation in the level of O2. The CO2 level in the confinement area did not exceed 0.45% at any time, with the average deviation in concentration of 0.23% during all confinement periods. The participants had portable ambient O2 concentration analyzers (Rae PGM-1100) in close proximity at all times during the hypoxic campaigns. The analyzers activated a safety audible alarm, if the O2 level decreased below the preset level.

Diet.

The participants received an individually tailored, strictly controlled, and standardized diet throughout all three phases of each campaign. Individualized caloric requirements were estimated using the modified Harris-Benedict resting metabolic rate equation (11) and subsequently multiplied by a physical activity level factor of 1.4 for the ambulatory (Pre phase, Post phase, and HAMB confinement) and of 1.2 for the bed-rest phases (NBR and HBR confinement). The targeted daily composition for dietary fat intake was 30% of the total energy intake, whereas protein intake was aimed at 1.2 g/kg. Accordingly, the targeted baseline macronutrient composition (expressed as a percentage of total dietary energy intake) was ∼55% of carbohydrates, ∼30% of fat, and ∼15% of protein. The intake of sodium was targeted to be <3,500 mg/day. The participants were encouraged to maintain their minimal daily fluid intake at 28.5 ml/kg. They were supplemented with vitamin D3 (1,000 IU/day) throughout all campaigns and were not allowed to eat or drink anything outside of the provided menu, including alcohol or caffeine-containing beverages.

A 14-day menu was prepared before the first campaign and rotated throughout the campaign duration. The same daily menu was used during the subsequent two campaigns to ensure that the participants consumed identical meals on the same days of each respective campaign. The menu was designed using the web-based application Open Platform for Clinical Nutrition (OPKP, Jozef Stefan Institute, Ljubljana, Slovenia). Five daily meals (breakfast, morning snack, lunch, afternoon snack, and dinner) were always served at the same time of the day throughout the campaigns. Each food item was weighed on a precision (+0.1 g) scale (TPT 6C, Libela ELSI, Celje, Slovenia) connected to a custom-developed, computer-based food recording and analysis system (Piki 2.0, Faculty of Computer Science, University of Ljubljana, Ljubljana, Slovenia). In case of any leftovers, the unconsumed food items were reweighed, and the value was deducted from the initial weight to provide the actual food intake. The Piki 2.0 system enabled real-time monitoring of the daily energy, macronutrient, and fluid intake for each individual.

Water balance.

Water balance was calculated daily during each confinement period by deducting the total daily water output from the total daily water input. It was assumed that, for every 100 g of consumed carbohydrate, protein, and fat, the resulting metabolic water production is 55, 41, and 107 g, respectively (1). The total daily water output included urinary output and insensible water loss. Urine was collected daily to obtain 24-h pools. Each urine collection started at 07:00 AM and was performed for the ensuing 24 h. Insensible water loss was estimated by assuming that, for every 1,000 kcal of energy consumed, 430 ml of water were lost as a consequence of respiration and skin perspiration (1).

All measurements were taken after an overnight fast at 07:30 AM. Subjects in the HAMB trials were requested not to get out of bed before these measurements.

Ophthalmological assessment.

With the exception of the measurements of IOP, which were conducted in the morning at the Olympic Sport Centre Planica, all ophthalmological assessments were conducted at the Eye Hospital of the University Medical Centre Ljubljana. Baseline measurements were determined during the initial medical screening conducted on the subjects, approximately 1 wk before the start of their first intervention. The remaining measurements were conducted on the last day of the prevailing intervention (day 10). Participants were transported to the Eye Hospital by ambulance. The travel time to the Eye Hospital was ∼1.5 h. Subjects in the bed-rest conditions (NBR and HBR) were transported in the horizontal position and remained in this position for the entire period. The exception was a 5-min period during which the OCT measurements were conducted. For these measurements, the subjects were allowed to rest on their elbows, as was allowed during eating (see above). Subjects in the hypoxic condition (HBR and HAMB) were requested to breathe a hypoxic mixture similar to that in the hypoxic facility of the Olympic Sport Centre Planica, for the duration of the travel to and from the Eye Hospital and during the OCT tests. The calibrated hypoxic gas had an oxygen fraction (Fo2) of 0.135, and a nitrogen fraction (Fn2) of 0.865. The breathing gas was delivered from compressed gas cylinders to a meteorological balloon. Subjects inspired the hypoxic gas mixture from the meteorological balloon via a close-fitting oronasal mask (Hans Rudolf, Kansas City, MO). In the hypoxic trials, subjects wore a pulse oximeter at all times, to ensure that they maintained the same oxygen saturation as in the Olympic Sport Centre Planica, thus ensuring that no leakage of gas occurred while breathing through the oronasal mask. During the transfer of subjects by ambulance to the Eye Hospital, the SpO2 in the HBR and HAMB trials remained stable and similar to the levels observed in the hypoxic facility of the Olympic Sport Centre. Pairs of subjects were accompanied in the ambulance by two nurses and an engineer. The former were responsible for the well-being of the subjects during the transfer, and the latter for the breathing gas mixtures.

On completion of the OCT measurements for the prevailing conditions (NBR, HBR, HAMB), subjects were switched to a breathing mixture that contained the same Fo2, but contained an elevated fraction of carbon dioxide (Fco2). CO2 in this breathing mixture was 0.01 (i.e. 1% by volume). This was to simulate the acute effect of a hypercapnic mixture, as experienced by astronauts on board the ISS, on the retinal layers. Thus subjects in the HBR and HAMB conditions breathed a hypoxic hypercapnic mixture (Fo2 = 0.135; Fco2 = 0.01; Fn2 = 0.855) during this 15-min period (10 min prebreathing, followed by 5 min during the OCT measurements), and subjects in the NBR condition inspired a normoxic hypercapnic mixture (Fo2 = 0.2093; Fco2 = 0.01; Fn2 = 0.7807). Following a 10-min period of breathing this mixture, subjects' posterior part of the eyes were again assessed with OCT.

Measurement of IOP.

IOP was measured with a Pulsair IntelliPuff noncontact tonometer (Halma India Pvt., Mumbai, India) several days before the onset of the interventions and on the last day of the intervention. The measurements were conducted in the morning, with the subject in the supine position.

Measurement of retina and choroid.

OCT is routinely used to assess retinal thickness and the thickness of individual retinal layers in a quantitative and reproducible manner. Recent advances in OCT technology have allowed enhanced visualization of the choroidal anatomy (37), enabling detailed evaluation of the choroid (22, 27).

OCT scans were obtained with a Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany), with viewing module version 5.1.2.0 (3.9-mm axial resolution) using enhanced depth imaging. All images were recorded at the same time of the day, between 12:00 AM and 2:00 PM. A circular 360° scan centered on the optic disk (standardized diameter: 3.4 mm) was obtained on each occasion. The RNFL thickness (from internal limiting membrane inner margin to RNFL layer outer margin) and retinal thickness (from internal limiting membrane inner margin to the outer edge of the RPE) were automatically segmented using the HEYEX software interface (version 1.6.2.0; Heidelberg Engineering). The software automatically overlays a six-sector circular grid centered on the optic nerve, and the retinal thickness is then determined for each sector of the grid. Our analysis included the values of all six sectors. For choroidal thickness (from the outer edge of the RPE to the inner chorioscleral interface), the segmentation line was moved manually to fit the inner scleral border by an examiner who was masked to the subjects' experimental conditions (Fig. 1).

Optical coherence tomograph of the optic disk from a healthy individual. The exact location of the scan is shown on a corresponding infrared reflectance image (top image). The location of the large retinal vessels and the retinal capillary microvasculature is highlighted in white. The location of choroidal vasculature is shown with small (choriocapillaries) and large circles (larger choroidal vessels). The thickness of the retina, retinal nerve fiber layer (RNFL), and choroid is also highlighted.

Follow-up function with Spectralis OCT software was used to ensure measurements of the same position at different examinations. The reported measurements represented the average RNFL and retinal and choroidal thickness of each scan.

Statistical analysis.

To examine the changes among the experimental conditions (NBR, HBR, HAMB), the repeated ANOVA was used for each of the examined variables (choroid, retina, RNFL). In cases where the assumptions of sphericity were not met in the within-subjects repeated-measures analyses, based on the results of Mauchly's test of sphericity, the Green-House Geisser correction and the corresponding degrees of freedom were applied for subsequent F-statistic estimation (9, 31). Bonferroni-corrected t-tests followed any significant effects in the ANOVA models testing pairwise comparisons (31). The significance level was set at P < 0.05. The Statistical Package for Social Sciences (SPSS 22.0 Win) was used for all analyses.

RESULTS

All seven subjects successfully completed the three experimental trials. One subject experienced AMS during the first day of the hypoxic interventions (HAMB and HBR). A second subject also experienced AMS during the first day of the hypoxic exposure in the HBR condition. In these cases, the subjects were transferred to a room where the Fo2 was higher, simulating an altitude of 3,000 m. In both subjects, the symptoms of AMS resolved as soon as they were removed from the area where Fo2 was at a level simulating the target altitude of 4,000 m. The subjects remained at a simulated altitude of 3,000 m overnight and continued their hypoxic exposure at the target altitude of 4,000 m the following day (day 2).

With the exception of SpO2, which was maintained ∼10% lower in the hypoxic (HBR and HAMB) trials compared with the normoxic trials (NBR), there were no major differences in the morning measurements of HR or arterial pressure (Table 2).

SpO2, HR, SAP, DAP, and IOP obtained in the fasted supine state at 07:30 AM 1 day before the intervention and on day 10 of the intervention

IOP.

There was no significant difference in the IOP measured at baseline and at the end of the three interventions (Table 2). There were also no significant differences in IOP between the NBR, HBR, and HAMB conditions.

Thickness of the posterior eye layers (RNFL, retina, choroid) 5 days before the interventions and on day 10 of the NBR, HBR, and HAMB trials

Retina.

No significant differences were observed between the baseline, NBR, HBR, and HAMB measures (Table 3). There was also no significant difference in the overall retinal thickness between the normocapnic and hypercapnic trials in the NBR (P = 0.383), HBR (P = 0.645), and HAMB (P = 0.929) conditions.

DISCUSSION

The principal finding of the present study is that a 10-day exposure to hypoxia, irrespective of physical activity (HBR and HAMB), significantly increased the thickness of the RNFL. In contrast, the thickness of the choroid was not affected by the hypoxia, but by the bed-rest conditions (NBR and HBR). The addition of 1% CO2 to the breathing mixture caused a significant increase in the thickness of the RNFL only in the NBR condition, but did not cause any further increases in the thickness of the RNFL in the HBR and HAMB conditions. These results indicate that the increases in the thickness of the RNFL is mainly mediated by hypoxia and are not augmented by hypercapnia. It may also suggest that the effect of hypercapnia and hypoxia on RNFL are similar. In contrast, the thickness of the choroid is influenced by bed rest and not by hypoxia. The following discussion will, therefore, focus on the vasoactive effect of hypoxia on the RNFL and the hydrostatic effect, arising most likely from the cephalad displacement of body fluids, on the choroid.

IOP.

In contrast to the observations by Chiquet et al. (2) of a decrease in IOP following a 7-day bed rest, we observed no change in IOP (Table 2). The difference in these results may be attributed to the hydrostatic effect, since the bed-rest protocol utilized by Chiquet et al. (2) incorporated a 6° head-down tilt. Drozdova and Nesterenko (5) reported a decrease in IOP during a horizontal bed-rest protocol, but the duration was 70 days. Thus our results are not necessarily contrary to these observations, since we utilized a horizontal bed rest of only a 10-day duration.

Retinal and choroidal vessels.

Embryologically, the retina is an extension of the diencephalon, and both organs share a similar pattern of vascularization during development (4, 28). There is a close anatomical correlation between both the macrovascular and the microvascular blood supply to the brain and the retina, and both vascular networks share similar vascular regulatory processes (3, 10). The retina receives its nutrients from two separate circulations: the retinal and the choroidal circulation. Although the retinal and choroidal vessels are all derived from the ophthalmic artery, a branch of the internal carotid, they differ morphologically and functionally. To understand the different responses of the vessels in the choroid and retina layers, it is also necessary to review the manner of their regulation (26).

The retinal capillary microvasculature has two distinct beds: the larger vessels and the superficial capillary layer in the RNFL, and the deeper capillary layer extending into the inner nuclear and outer plexiform layers (Fig. 1). In the peripapillary area, an additional capillary network lies in the superficial portion of the RNFL, constituting the radial peripapillary capillaries (12). The changes we observed in the RNFL thickness most likely reflect the changes in the diameter of retinal capillaries. The intraocular part of the central retinal artery, including retinal circulation, is without autononomic innervation (8, 13, 16, 38), and, therefore, its regulatory control mechanisms are not under neurogenic control. Despite this, there is efficient autoregulation, mainly influenced by local factors, including mediators released by endothelial cells and surrounding retinal tissue (3). Retinal vessel tone thus relies predominantly on local vascular control mechanisms and is, among others, related to the modifications of the arterial blood gases. Thus hypoxia (7) and hypercapnia (36) result in significant increases in retinal arteriolar diameter. Our results support the vasoactive effect of hypoxia and demonstrate that acute hypercapnia elicits a significant increase in the thickness of the RNFL only in the NBR condition. It may be that the actions of hypoxia and hypercapnia are similar and not additive or synergistic; thus the combined effect is not evident in the HBR and HAMB conditions, but similar to those of the NBR condition.

The choroid is arranged in layers of vessels, from larger in the outer part toward smaller and choriocapillaries in the inner part, with no distinct border between these layers (Fig. 1). Choroidal capillaries are large in diameter compared with retinal capillaries and provide low resistance to blood flow. They also have fenestrations, which do not exist in the retinal capillaries, so they readily leak plasma proteins responsible for maintaining the interstitial fluid compartment. In contrast to the retinal circulation, the choroidal circulation is mainly controlled by sympathetic innervation and is not autoregulated (3). The peripapillary choroid has an important role in the blood supply of the anterior optic nerve, including the optic disk (14).

The increase in the thickness of the choroid observed in the present study supports the hypothesis that the spaceflight-induced cephalic shift of body fluids contributes to the etiology of MOS. Indeed, the increase in the choroid layer thickness could contribute to the hyperopic shift, by reducing the optical length between the central retinal region and the anterior ocular structures.

MOS on the ISS and in future habitats.

MOS was discovered in astronauts following longer missions on board the ISS. The environment on the ISS is normoxic, but hypercapnic. In addition, the astronauts follow a strict regimen of daily exercise to prevent loss of muscle and bone mass. This exercise is conducted in an atmosphere with high concentrations of CO2, but our results indicate that acute hypercapnia does not cause a change in the thicknesses of the retinal layers. It may be that the exercise training on the ISS, akin to training in a 6° head-down tilt position on Earth, causes an increase in intracranial pressure, which may impact on the choroidal vessels and IOP, leading to the structural and functional changes of the posterior eye. Assuming that future hypobaric hypoxic habitats will also have an inadequate CO2 scrubbing system as the ISS, then the thickness of the RNFL layer will be enlarged, as a consequence of local autoregulation of the vessels. Since there is some gravity on the moon and Mars, the hydrostatic-induced changes in the choroid will most likely not be as great as observed in the present study.

Blood-brain barrier.

A possible window into the health of the brain's vascular network is provided by the closely related retinal blood vessels of the eye. Retinal and cerebral small vessels share similar embryological origin, as well as structural and physiological features (24). Thus, assessing retinal vasculature may provide a useful noninvasive method to visualize the state of the brain's microcirculation in vivo.

The theory put forward by Taylor et al. (34), based on experiments conducted on mice on board the ISS, proposes that the hypercapnia of the environment within the ISS compromises the integrity of the blood-brain barrier (17), thus allowing factors that affect the peripheral circulation to also affect the cerebral circulation. The mice in the study of Taylor et al. (34) were exposed to the ISS environment for a total of 13 days, during which the Pco2 ranged between 2.0 and 3.0 Torr for the majority of the time, with a spike toward the end of the mission attaining 3.5 Torr. In the present study, the hypercapnic exposure was acute, as the subjects only inspired the hypercapnic mixture for a total of 10 min before the OCT examination and up to 5 min during the OCT examination. Nevertheless, there was no indication that hypercapnia caused any change in the retinal thickness, which would be changed, if the blood-retinal barrier were compromised, in the hypercapnic trials of the normoxic (NBR) and hypoxic (HBR and HAMB) conditions, suggesting no evidence of any effect on the blood-brain barrier.

Hypoxic and exertion-induced retinopathy.

During high-altitude expeditions, it is common to observe retinopathy as a consequence of the hypoxia: the latter increases the permeability of the retinal vessels, causing leakage into the retina and engorgement of the retinal blood vessels (15). Exertional exercise, which markedly elevates systemic arterial pressure, may also cause leakage of blood into the retina. The level of hypoxia in the HBR and HAMB trials was not of such magnitude as to be able to cause high-altitude retinopathy. Furthermore, the exercise intensity in the HAMB condition was maintained such that the HR was equivalent to that observed at ∼50% of the hypoxic PPO; thus exertional retinopathy was also unlikely. Any hypoxia- and/or exercise-induced effect on intracranial pressure would also have been reflected in the IOP, which remained constant at normal levels throughout the three interventions.

Methodological considerations.

To avoid significant diurnal changes of choroidal thickness (35), our measurements were performed at the same time of day, between 12:00 AM and 2:00 PM. Usui et al. (35) reported no significant correlation between choroidal thickness and diastolic arterial pressure, HR, and IOP, but there was a significant negative correlation with mean systolic arterial pressure. Since there were no differences in the morning measurements of arterial pressure (systolic and diastolic arterial pressure), IOP, and HR, it is unlikely that these factors contributed to the differences observed in the thicknesses of the retinal layers and choroid in the three conditions.

Subjects were not assigned randomly to all the conditions (i.e. NBR, HBR, and HAMB) of the study, but only to the first condition. Thereafter, the order of the conditions for each subject pair was balanced according to a Latin square design. This type of approach should have minimized any order effect.

Conclusions.

The choroid layer was affected solely by the hydrostatic factor, since only the differences between the bed rest (NBR and HBR) and ambulatory (HAMB) measurements were significant. In contrast, the RNFL was affected predominantly by the hypoxic stimuli, as evidenced by the significant differences between the normocapnic baseline measurements and those obtained in the HBR and HAMB trials. The values observed in the NBR and HBR conditions were also significantly different. Since a significance was also observed between the HBR and HAMB conditions, it may be concluded that RNFL is predominantly affected by the hypoxia, but is also to some degree affected by posture. Noteworthy is also the significant effect of the hypercapnic stimulus in the NBR, but not the HBR and HAMB conditions. This would suggest absence of a synergistic response between hypoxia and hypercapnia.

GRANTS

This work was funded by the European Space Agency (ESA) Programme for European Cooperating States (PECS; ESA Contract No. 4000111808/14/NL/NDe), and the Swedish National Space Board No 109/11:2.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

ACKNOWLEDGMENTS

We are indebted to the subjects for dedication to the project, to the medical staff of the PlanHab study for assistance during the transport to the Eye Hospital, and to Eva Kumprej and Bogomir Vrhovec for excellent technical support. We are also very grateful to Dr. Nektarios Stavrou (Faculty of Physical Education & Sport Science, National & Kapodistrian University of Athens, Athens, Greece) for kind assistance and support with the statistical analyses.

. Proceedings of the ESA Topical Team Workshop on simulation of Lunar Habitats. In: Book of Abstracts of the 36th Annual Meeting of the International Society for Gravitational Physiology. Ljubljana, Slovenia: Studio Print, 2015, p. 133–139.